Thiourea and urea liquid-phase combinatorial libraries: synthesis and apoptosis induction

ABSTRACT

This invention provides combinatorial chemistry libraries containing thiourea and urea compounds. In addition, the invention relates to methods for constructing combinatorial chemistry libraries containing thiourea and urea compounds. Furthermore, this invention relates to methods for the identification of bioactive thiourea and urea compounds as well as compositions and therapeutic methods for treating cancer.

PRIORITY OF THE INVENTION

This application is a continuation application of internationalapplication number PCT/US00/06989 filed on 19 Mar. 2000 claimingpriority under 35 U.S.C. 119 (a)-(e) to U.S. Provisional ApplicationNumber 60/125,146 filed on 19 Mar. 1999; the international applicationwas published under PCT Article 21(2) in English as WO 00/56681.

FIELD OF THE INVENTION

This invention relates to combinatorial chemistry libraries containingthiourea and urea compounds. In addition, the invention relates tomethods for constructing combinatorial chemistry libraries containingthiourea and urea compounds. Furthermore, this invention relates tomethods for the identification of bioactive thiourea and urea compoundsas well as compositions and therapeutic methods for treating cancer.

BACKGROUND OF THE INVENTION

A common method of drug discovery is to first delineate a biochemicalpathway that is involved in a targeted pathological process. Thebiological pathway is analyzed so as to determine crucial elementswhich, if obstructed, restrained or otherwise adversely modified couldinhibit the pathological process. Generally, an assay can be developedthat is indicative of the functional ability of an element of thebiochemical pathway. The assay can then be performed in the presence ofa number of different molecules. The researcher can then determine themolecules that have the desired effect on the pathway, and that moleculeor molecules can be used in treatment or can be further modified toaugment and enhance the desired effect.

As the assays that are indicative of these pathways become faster, andmore easily automated, the rate determining step regarding molecularscreening becomes the production of the molecules to be tested. Thus,the development of techniques to rapidly and systematically synthesizelarge numbers of molecules possessing diverse structural properties hasgrown in importance. On such technique for rapidly and systematicallysynthesizing large numbers of molecules possessing diverse structuralproperties is the construction of combinatorial libraries. Combinatorialchemistry employing solution-phase combinatorial synthesis plays andincreasingly important role in drug discovery efforts.

Combinatorial libraries are typically formed via a multistep syntheticprocedure employing either solution-phase or solid-phase methods. Theprocedure typically includes mixtures of different subunits which areadded stepwise to growing oligomers until a desired oligomer size isreached. Alternatively, the subunits can be combined in one syntheticstep to produce a random array of oligomers or a combination of the twoprocedures may be employed. The result is the rapid synthesis of alarge, diverse group of chemical compounds that can be screened with thepredictive assay developed with regard to the targeted pathologicalprocess. Since the chance of finding useful molecules increases with thesize of the combinatorial library, it is desirable to generate librariescomposed of large numbers of oligomers which vary in their subunitsequence.

Apoptosis is a biochemical process that is an important part of a numberof diseases. Apoptosis is a common mode of eukaryotic cell death whichis triggered by an inducible cascade of biochemical events leading toactivation of endonucleases that cleave the nuclear DNA intooligonucleosome-length fragments. Several of the biochemical events thatcontribute to apoptotic cell death as well as both positive and negativeregulators of apoptosis have recently been identified (Whyllie A., etal. (1980) Int. Rev. Cytol. 68, 251-305; Steller H., (1995) Science 267,1445-1449; Fraser, A., Evan, G. (1996) Cell 85, 781-784; and Korsmeyer,S. J. (1995). Trends Genet. 11, 101-105). Apoptosis plays a pivotal rolein the development and maintenance of a functional immune system byensuring the timely self-destruction of autoreactive immature and maturelymphocytes as well as any emerging target neoplastic cells by cytotoxicT cells.

In addition to the beneficial effects associated with apoptosis,inappropriate apoptosis contributes to the pathogenesis and drugresistance of human leukemias and lymphomas (Cohen, J. J., et al.(1992)Annu. Rev. Immunol. 10, 267-293; Linette, G. P., Korsmeyer, S. J.(1994) Curr. Opin. Cell Biol. 6, 809-815; and Thompson, C. B.(1995)Science 367, 1456-1462). Thus, agents that are useful to modulateapoptosis are potentially useful as therapeutic agents for treatingdiseases in which inappropriate apoptosis is implicated. As a result,there is a considerable amount of ongoing research devoted to theidentification of molecular regulators of apoptosis, and there iscurrently a need for novel agents (e.g. chemical or biological), andnovel therapeutic methods, that are useful for modulating apoptosis.Such agents and methods may be useful for treating cancer (e.g.leukemias and lymphomas) or immune disorders in mammals. They may alsobe useful as pharmacological tooIs for use in in vitro or in vivostudies to enhance the understanding of the molecular basis of apoptosis(e.g. the pro-apoptotic versus the anti-apoptotic regulatory signal), aswell as the pathogenesis of human lymphoid malignancies.

Novel thiourea and urea compounds have been found to be potent cytotoxicagents with potent activity against cancer cells. For example, certainthiourea and urea compounds exhibit potent cytotoxic activity,particularly against human leukemic cell lines. Additionally, thioureaand urea compounds have been found to be nonnucleoside inhibitors of HIVreverse transcriptase. Currently the production of thiourea and ureacompounds however, is limited to the small scale synthesis of individualmolecules. Thus, a method for the rapid and systematic synthesis oflarge numbers of thiourea and urea compounds possessing diversestructural properties is desirable.

SUMMARY OF THE INVENTION

Generally, the present invention relates to a combinatorial libraryincluding compounds of the Formula I

wherein X is S or O;

-   -   R and R₁ are individually        where Ar is aryl; R₂ is H or C₁ to C₆ alkyl; n is 0-3 and where        the aryl moiety is optionally substituted from 1 to 7 times with        any combination of H, halo, alkyl, haloalkyl, arylalkyl, alkoxy,        haloalkoxy, and aralkoxy. One embodiment is a combinatorial        library of claim 1, wherein R and R₁ are individually        where R₂ is H or C₁ to C₆ alkyl; n is 0-3 and where the phenyl        moiety is optionally substituted from 1 to 5 times with any        combination of R₃ R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃,        R₁₄, R₁₅, R₁₆, R₁₇, R₁₈, R₁₉, R₂₀, R₂₁, R₂₂, R₂₃, R₂₄, R₂₅, R₂₆,        and R₂₇; and where R₃ is H, R₄ is 2-methyl, R₅ is        2-trifluoromethyl, R₆ is 2-fluoro, R₇ 2chloro, R₈ is 2-methoxy,        R₉ is 2-ethoxy, R₁₀ 3-methyl, R₁₁ is 3-trifluoromethyl, R₁₂ is        3-fluoro, R₁₃ is 3-chloro, R₁₄ is 3-iodo, R₁₅ is 3-methoxy, R₁₆        is 4-methyl, R₁₇ is 4-trifluoromethyl, R₁₈ is 4-fluoro, R₁₉ is        4-chloro, R₂₀ is 4-bromo, R₂₁ is 4-methoxy, R₂₂ is        5-trifluoromethyl, R₂₃ is 5-fluoro, R₂₄ is 6-fluoro, R₂₅ is        5-methoxy, R₂₆ is 3-benzyloxy, and R₂₇ is 4-benzyloxy.

Another embodiment is a method for synthesizing a combinatorial libraryincluding compounds of the Formula I:

where X is S or O;

-   -   R and R₁ are individually        where Ar is aryl; R₂ is H or C₁ to C₆ alkyl; n is 0-3 and where        the aryl moiety is optionally substituted from 1 to 7 times with        any combination of H, halo, alkyl, haloalkyl, arylalkyl, alkoxy,        haloalkoxy, and aralkoxy, including the step of contacting a        subunit selected from the group consisting of urea and thiourea        with an amine in a suitable carrier.

Yet another embodiment is composition for determining possible apoptosisinduction agents for a biological substrate, comprising a combinatoriallibrary or compounds generated therefrom.

A further embodiment of the present invention is a method of killing acancer cell by contacting the cancer cell with a combinatorial libraryor compounds generated therefrom.

Another embodiment of the invention includes a kit for determiningpossible apoptosis induction agents for a biological substrate.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The Figures and the detailed description which follow moreparticularly exemplify these embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be more completely understood in consideration of thefollowing detailed description of various embodiments of the inventionin connection with the accompanying drawings, in which:

FIG. 1A and B: are FAB mass spectrum of Combinatorial Library 1.

FIG. 2A and B: are ESI mass spectrum (FIG. 4A) and a computer-generatedMS spectrum (FIG. 4B) of Combinatorial Library 34.

FIG. 3A and B: are ESI mass spectrum of CL35 (FIG. 3A) and a computergenerated mass spectrum (FIG. 3B) of CL35 for comparison.

While the invention is amenable to various modifications and alternativeforms, specifics thereof have been shown by way of example in thedrawings and will be described in detail. It should be understood,however, that the intention is not to limit the invention to theparticular embodiments described. On the contrary, the intention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is believed to be applicable to combinatorialchemistry libraries containing thiourea and urea compounds. Inparticular, the present invention is directed to combinatorial chemistrylibraries containing thiourea and urea compounds, methods forconstructing these libraries and methods for the identification ofbioactive thiourea and urea compounds. While the present invention isnot so limited, an appreciation of various aspects of the invention willbe gained through a discussion of the examples provided below.

The term “combinatorial library” refers to an intentionally createdcollection of differing molecules which can be prepared syntheticallyand screened for biological activity. A combinatorial library consistsof at least two compounds.

The term “combinatorial chemistry” refers to the synthesis of compoundsfrom sets of subunit and chemical reactions used in one or more reactionsteps.

The term “alkyl” refers to straight or branched hydrocarbon radicals,such as methyl, ethyl, propyl, butyl, octyl, isopropyl, tert-butyl,sec-pentyl, and the like. Alkyl groups can either be unsubstituted orsubstituted with one or more substituents, e.g., halogen, alkoxy, aryl,arylalkyl, aralkoxy and the like. Typically, alkyl groups include 1 to 8carbon atoms, preferably 1 to 5, and more preferably 1 to 3 carbonatoms.

The term “halo” refers to fluoride, chloride, bromide, and iodideradicals.

The term “aryl” refers to monovalent unsaturated aromatic carbocyclicradicals having a single ring, such as phenyl, or multiple condensedrings, such as naphthyl or anthryl, which can be optionally substitutedby substituents such as halogen, alkyl, arylalkyl, alkoxy, aralkoxy, andthe like.

The term “haloalkyl” refers to an alkyl group substituted with a haloradical as defined above.

The term “alkoxy” refers to an oxygen atom substituted with an alkylradical as defined above. Typical alkoxy groups include methoxy, ethoxy,propoxy, iopropoxy, and the like. Preferable alkoxy groups includemethoxy and ethoxy.

The term “arylalkyl” refers to an alkyl radical defined as abovesubstituted with an aryl radical as defined above. Typical arylalkylgroups include phenethyl, benzyl, and naphthethyl. Preferable alylalkylgroups include phenethyl and benzyl.

The term “aralkoxy” refers to an alkoxy group as defined above where thealkyl group is substituted with an aryl radical as defined above.

The term “haloalkoxy” refers to an alkoxy group as defined above wherethe alkyl group is substituted with a halo radical as defined above.

The term bioactive refers to a molecule that exhibits anti-cancer,anti-microbial, or anti-viral activity.

Thiourea and Urea Combinatorial Libraries

The present invention provides combinatorial libraries that includethiourea and urea compounds represented by the Formula I:

where X is S or O;

-   -   R and R₁ are individually        where Ar is aryl; R₂ is H or C₁ to C₆ alkyl; n is 0-3 and where        the aryl moiety is optionally substituted from 1 to 7 times with        any combination of H, halo, alkyl, haloalkyl, arylalkyl, alkoxy,        haloalkoxy, and aralkoxy. In addition, R and R₁ include

In one embodiment R₁ and R₂ are individually

where R₂ is H or C₁ to C₆ alkyl; n is 0-3 and where the phenyl moiety isoptionally substituted from 1 to 5 times with any combination of R₃ R₄,R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, R₁₈, R₁₉,R₂₀, R₂₁, R₂₂, R₂₃, R₂₄, R₂₅, R₂₆, and R₂₇; and where R₃ is H, R₄ is2-methyl, R₅ is 2-trifluoromethyl, R₆ is 2-fluoro, R₇ 2-chloro, R₈ is2-methoxy, R₉ is 2-ethoxy, R₁₀ 3-methyl, R₁₁ is 3-trifluoromethyl, R₁₂is 3-fluoro, R₁₃ is 3-chloro, R₁₄ is 3-iodo, R₁₅ is 3-methoxy, R₁₆ is4-methyl, R₁₇ is 4-trifluoromethyl, R₁₈ is 4-fluoro, R₁₉ is 4-chloro,R₂₀ is 4-bromo, R₂₁ is 4-methoxy, R₂₂ is 5-trifluoromethyl, R₂₃ is5-fluoro, R₂₄ is 6-fluoro, R₂₅ is 5-methoxy, R₂₆ is 3-benzyloxy, and R₂₇is 4-benzyloxy.

In a preferred embodiment the combinatorial libraries include compoundsof Formula I where R₁ and R₂ are independently selected from the group

Combinatorial Synthesis

Combinatorial library synthesis is typically performed ether on a solidsupport, such as peptide synthesis resins, or in liquid phase. For solidsupport synthesis of combinatorial libraries a large number of beads orparticles are suspended in a suitable carrier, such as a solvent, in aninitial reaction vessel. The beads, for example, are provided with afunctionalized point of attachment for a chemical subunit. The beads arethen divided and placed in various separate reaction vessels. The firstchemical subunit is attached to the bead, providing a variety ofdifferently substituted solid supports. The beads are washed to removeexcess reagents and recombined. The beads are again divided intoseparate reaction vessels and the second chemical subunit is coupled tothe chemical module. This recombining and division synthetic process canbe repeated until each of a number of selected chemical subunits havebeen incorporated onto the molecule attached to the solid support.

Solid-phase synthesis makes it easier to conduct multistep reactions andto drive reactions to completion, because excess reagents can be addedand then easily washed away after each reaction step. But a much widerrange of organic reactions is available for liquid-phase synthesis, thetechnology used traditionally by most synthetic organic chemists, andproducts in solution can be more easily identified and characterized.Liquid phase synthesis of combinatorial libraries typically involvescombining all the desired chemical subunits in a suitable carrier, suchas a solvent, and applying reactions conditions which facilitate thecombining of the various chemical subunits in a random fashion toproduce an array of final molecules. Alternatively, the total number ofchemical subunits can be split into various grouping. These groupingscan be added stepwise to the reaction vessel containing a suitablecarrier and another grouping of chemical subunits thereby producing anarray of final molecules in a less-random, more systematic andcontrolled fashion. Thus, the synthesis of compounds of a combinatoriallibrary can take place through several sequential reaction steps inwhich the same or different sets of subunits and chemical reactions areused, as well as the reaction of multiple subunits in one reaction stepto form multicomponent compounds.

Combinatorial library synthesis can be performed either manually orthrough the use of an automated process. For the manual construction ofa combinatorial library, an individual would perform the cariouschemical manipulations. For the construction of a combinatorial librarythrough an automated process, the various chemical manipulations willtypically be performed robotically.

Combinatorial library synthesis of the present invention is typicallyperformed in liquid phase. The desired chemical subunits (e.g.thioureas, ureas, and amine as defined below) are combined and contactedwith each other in a suitable carrier, such as a solvent, at a suitabletemperature. Examples of suitable solvents include ethers, such asdiethyl ether (Et₂O), chlorinated solvents, such as methylene chloride(CH₂Cl₂), chloroform, or dichloroethane, aromatics, such as toluene, oracetonitrile. Preferably, the organic solvent is acetonitrile. Suitablereaction temperatures are those which allow the desired reaction toproceed to completion in a minimum amount of time while producing thedesired products in high yield and purity. Typically, reactiontemperatures are such that the carrier used refluxes. The resultingcrude reaction mixture is concentrated, diluted in a suitable solvent,such as a halogenated solvent, washed with an aqueous acid solution andthen neutralized. The crude reaction mixture can be concentrated by avariety of methods known to those of skill in the art such asdistillation, or evaporation at elevated temperatures or reducedpressure or both. Suitable halogenated solvents used to dilute theconcentrated crude reaction mixture include, for example, CH₂Cl₂,chloroform, and dichloromethane. Preferable halogenated solvents includechloroform. Aqueous acid solutions useful in the present invention areknown to those of skill in the art and include, for example, aqueoussolutions of hydrochloric acid or acetic acid. Neutralization agentsuseful in the present invention are known to those of skill in the artand include, for example, alkaline salts such as CaO, NaOH, KOH andNaHCO₃.

The present method typically employs a molar ratio of the variouschemical subunits of about 1:1. It shall be understood however, that themolar ratio among the various chemical subunits can be varied to as toaffect the final composition of the desired combinatorial library. Forexample, one subgroup may be added in excess so as to produce acombinatorial library with an excess of compounds which include thatparticular subgroup.

Typical chemical subunits include ureas and thioureas. Preferable ureasand thioureas include, for example, those that undergo alkylamino,arylamino, and arylalkylamino de-amination. Examples of most preferredureas and thioureas include1,1′-thiocarbonyldiimidizole (TCDI), and1,1′-carbonyldiimidizole (CDI).

Additional chemical subunits include compounds of Formula II

where Ar is aryl; R₂ is H or C₁ to C₆ alkyl; n is 0-3 and where the arylmoiety is optionally substituted from 1 to 7 times with any combinationof H, halo, alkyl, haloalkyl, arylalkyl, alkoxy, haloalkoxy, andaralkoxy.

Preferred subunits include compounds of Formula III

where R₂ is H or C₁ to C₆ alkyl; n is 0-3 and where the phenyl moiety isoptionally substituted from 1 to 5 times with any combination of R₃ R₄,R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, R₁₈, R₁₉,R₂₀, R₂₁, R₂₂, R₂₃, R₂₄, R₂₅, R₂₆, and R₂₇; and where R₃ is H, R₄ is2-methyl, R₅ is 2-trifluoromethyl, R₆ is 2-fluoro, R₇ 2-chloro, R₈ is2-methoxy, R₉ is 2-ethoxy, R₁₀ 3-methyl, R₁₁ is 3-trifluoromethyl, R₁₂is 3-fluoro, R₁₃ is 3-chloro, R₁₄is 3-iodo, R₁₅ is 3-methoxy, R₁₆ is4-methyl, R₁₇ is 4-trifluoromethyl, R₁₈ is 4-fluoro, R₁₉ is 4-chloro,R₂₀ is 4-bromo, R₂₁ is 4-methoxy, R₂₂ is 5-trifluoromethyl, R₂₃ is5-fluoro, R₂₄ is 6-fluoro, R₂₅ is 5-methoxy, R₂₆ is 3-benzyloxy, and R₂₇is 4-benzyloxy.

Most preferred subunits include the following compounds

Screening

The present invention is directed toward the generation of thiourea andurea combinatorial libraries. These libraries can be used to select oneor more urea or thiourea species that demonstrate biological activitysuch as, for example, apoptotic activity. Apoptosis plays a pivotal rolein the development and maintenance of a functional immune system byensuring the timely self-destruction of autoreactive immature and maturelymphocytes as well as any emerging target neoplastic cells by cytotoxicT cells. In addition to the beneficial effects associated withapoptosis, inappropriate apoptosis contributes to the pathogenesis anddrug resistance of human leukemias and lymphomas.

Several methods have been developed to screen libraries of compounds toidentify those compounds having the desired biological activity. Suchmethods are well known to those of skill in the art. For example, acellular or enzyme solution may be combined with at solution of thecompounds of a particular combinatorial library under conditionsfavorable to elicit a biological response such as inhibition of anenzyme or regulation of a cellular pathway. The biological activity oflibrary compounds may be detected by any of the numerous biologicalassays which are well known in the art such as, for example, the MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay(Boehringer Mannheim Corp., Indianapolis, Ind.) or the inapoptosis/TUNEL assay (Zhu D et al., Clinical Cancer Research4:2967-2976, 1998). In cases where the compounds of a givencombinatorial library demonstrate biological activity by binding to abiological target such as an enzyme the compound/biological targetcomplex can be separated from other assay components using variousmethods known to those of skill in the art such as size-exclusionchromatography. The compound/biological target complex can then bedenatured to release the compound which can then be isolated usingvarious methods known to those of skill in the art, such as HPLC, andsubjected to mass spectrometry for identification.

An alternative manner of identifying biologically active compounds isiterative synthesis and screening to deconvolute the combinatoriallibrary. Iterative synthesis/screeing involves the synthesis ofcompounds in such a manner that a combinatorial library results that isnot directly resolvable to determine the identity of discretebiologically active compounds, but that instead is resolvable todetermine the identity of a specific compound in any mixture that showsbiological activity when assayed. A new sublibrary is then synthesizedbased on this information and assayed, and the identity of the nextspecific subunit determined. The iterative process is continued untilthe identity of a complete, active molecule is determined. Iterativesynthesis/screeing has several characteristics including diminishinglibrary size as the iterations proceed, ending with the last step whichinvolves the synthesis and assaying of the individual compound. Variousmethods of deconvolution fall within the iteration definition and willbe considered as a form of iterative synthesis/screening.

EXAMPLES Example 1 Synthesis of N,N′-Bis(2-phenylethyl)thioureaCombinatorial Libraries, Deconvolution and Identification ofBiologically Active Species

The reaction is accomplished by phenethylamine substitution ofimidazoles in thiocarbonyldiimidazole (TCDI) in solvent acetonitrileunder reflux. The reaction is carried out in equimolar ratio. After thereaction is completed, the substituted imidazole is protonated withdilute aqueous acid and separated during solvent/solvent extraction.

Stage (I): synthesis of compound 1 in Scheme 1.

The synthesis of compound 1 was carried out by adding an equimolaramount of p-chlorophenethylamine into a solution of TCDI in acetonitrileat 0° C., followed by reflux for 1 h. Thin-layer chromatography (TLC)indicated the completion of the reaction. The concentrated reactionmixture was re-dissolved in CHCl₃. The substituted imidazole from TCDIwas washed out with dilute hydrochloric acid (0.65 M) duringliquid/liquid extraction. After neutralization (saturated NaHCO₃solution), drying (MgSO₄) and concentration; a white powder (m.p. 126°C.) was obtained as the desired product (1) with 90% yield and 99%purity by HPLC.

The structure of compound 1 was confirmed with ¹H NMR, ¹³C NMR,MALDI-TOF MS, and elemental analysis:

1,3-Bisp-chlorophenethyl)-2-thiourea (compound 1). ¹H NMR (300 MHz,CDCl₃, TMS) d 7.28 (d, ³J(H,H)=8.4 Hz, 4H, Ar), 7.11 (d, ³J(H,H)=8.4 Hz,4H, Ar), 5.58 (bs, 2H, NH), 3.62 (bs, 4H, α-CH₂), 2.84 (t, ³J(H,H)=6.9Hz, 4H, β-CH₂); ¹³C NMR d 181.6, 136.6, 132.4, 130.0, 128.7, 45.2, 34.4;MS: m/z (MALDI-TOF) 353.3 (C₁₇H₁₈C₁₂N₂S requires 353.3); UV/Vis (CH₃CN)I_(max) 194, 224, and 249 nm; HPLC retention time 15.9 min with thefollowing conditions: HP 1100 Series with a LiChrospher 100 RP-18 (5 mm)column (Part #799250D-584, 250-4), mobile phase:water/acetonitrile=50/50, flow rate: 1.5 mL/min, injection volume: 30mL, wavelength: 225 nm. Anal. Calcd for C, 57.79; H, 5.13; Cl, 20.07; N,7.93; S, 9.07. Found: C, 57.90; H, 5.13; Cl, 20.02; N, 7.88; S, 9.03.

A two-step one-pot procedure was also developed to substitute oneimidazole on TCDI for each step. First, one molar equivalent ofp-chlorophenethylamine was allowed to react with TCDI under 0° C.followed by reflux for 1 h. The mono-substituted thiocarbonylintermediate was less polar as indicated by TLC (R_(f) 0.76) vs. thedi-substituted compound 2 (R_(f) 0.46). After the completion of thefirst substitution by p-chlorophenethylamine, one molar equivalent ofphenethylamine was added at the room temperature, followed by reflux for1 h. After work-up, a yellow wax was obtained as product with aquantitative yield.

The structure of compound 2 was confirmed with ¹H NMR, ¹³C NMR,MALDI-TOF MS and elemental analysis:

1-(p-Chlorophenethyl)-3-phenethyl-2-thiourea (Compound 2). ¹H NMR (300MHz, CDCl₃, TMS) d 7.33-7.09 (m, 9H, Ar), 5.62 and 5.55 (2 bs, 2, NH),3.62 (bs, 4H, α-CH₂), 2.87 (t, ³J(H,H)=6.9 Hz, 2H, β-CH₂), 2.83 (t,³J(H,H)=6.6 Hz, 2H, b-CH₂); ¹³C NMR d 181.5, 138.1, 136.7, 132.3, 130.0,128.7, 128.6, 126.6, 45.3, 35.0, 34.4; MS: m/z (MALDI-TOF) 320.5(C₁₇H₁₉CIN₂S requires 318.9); UV/Vis l_(max) 192, 212 (shoulder), and249 nm; HPLC retention time 10.3 min. Anal. Calcd for C₁₇H₁₉ClN₂S: C,64.04; H, 6.01; Cl, 11.12; N, 8.79; S, 10.06. Found: C, 64.00; H, 6.07;Cl, 11.00; N, 8.87; S, 10.11.

Stage (II): synthesis of a small mixture library (SML) containing onlythree members (1, 2 and 3 in Scheme 2) as a model, starting from anequimolar mixture of phenethylamine and p-chlorophenethylamine inacetonitrile. The same procedure was followed as in stage (I). Thelibrary was obtained as a white powder with a quantitative yield. SCHEME2 Synthesis of a small mixture library (SML)

Compound R R′ Ratio 1 H H 1 2 Cl Cl 1 3 H Cl 2

The composition of SML was confirmed with ¹H NMR, LC-MS and elementalanalysis:

SML. ¹H NMR (300 MHz, CDCl₃, TMS) d 7.35-7.08 (m, 9H, Ar), 5.62 (bs, 2H,NH), 3.62 (bs, 4H, α-CH₂), 2.84 (quintet, 4H, β-CH₂). LC-MS retentiontime 7.4 min corresponds to m/z 285.0 Da, 9.4 min to 319.0 Da, and 12.2min to 353.0 Da; calcd mass of 284.4 (3), 318.9 (2) and 353.3 (1). Anal.Calcd for 1/2/3=1/2/1: C, 64.42; H, 6.05; Cl, 10.58; N, 8.84; S, 10.11.Found: C, 64.06; H, 6.02; Cl, 11.10; N, 8.84; S, 10.08.

Stage (III), a main library (CL1) was synthesized (Chart 1) utilizingthe same method as that given in Stage II above, and started with anequimolar solution of 15 amines. The number of members in acombinatorial library can be predicted based on the following formulathat takes into account the number of differing substituents, and thenumber of possible positions. $\begin{matrix}{{{Number}\quad{of}\quad{members}\quad{in}\quad{library}} = {n + {C\left( {n,r} \right)}}} \\{= {n + {{n!}/\left\lbrack {{\left( {n - r} \right)!}X\quad{r!}} \right\rbrack}}} \\{{{Number}\quad{of}\quad{members}\quad{in}\quad{library}} = {{15 + {{15!}/\left\lbrack {{\left( {15 - 2} \right)!} \times {2!}} \right\rbrack}} = 120.}}\end{matrix}$The main library, CL1, that was synthesized had 15 possible substituentsat two different possible positions, leading to 120 members in thecombinatorial library.

The composition of CL1 was confirmed with ¹H NMR and MS:

CL1. ¹H NMR (300 MHz, CDCl₃, TMS) d 7.42-6.70 (m, rel. intensity 16,Ar), 5.75 (br, rel. intensity 3.5, NH), 5.12 (2 peaks, rel. intensity 1,OCH₂-Ph), 3.86-3.61 (m, rel. intensity 15, a-CH₂ and OCH₃), 2.84 (m,rel. intensity 10, b-CH₂), 2.32 (s, rel. intensity 1, CH₃-Ph).

FIG. 1A shows the FAB MS spectrum of CL1. The mass range 284-709corresponds to the 34 different molecular weights out of 120 members inCL1. To assist visual comparison, a theoretical mass spectrum of CL1 wasgenerated manually with a DeltaGraph Program (FIG. 1B, which displaysprimary peaks only (isotopes not considered.). The mass distribution cangenerally be viewed as three groups: 284-422, 497-566, and 709. The FABMS in FIG. 1A matches the theoretical mass spectrum profile in FIG. 1B.

Stage (4): deconvolution and re-synthesis were guided by an iterativescreening procedure during which the cytotoxic anti-cancer activities ofthe individual sub-libraries were measured against the human leukemiacell lines NALM-6 and MOLT-3 using MTT assays. In general, wheneverfeasible, an active library was evenly splitted into two sub-librariesand upon biologic testing the more active one of the two sub-librarieswas selected for further iteration. However, this is not always truewhen even-splitting was not possible or some special consideration. Forexample, library CL1 was discovered to be active and was primarily splitto two uneven libraries (CL3 with 55 members, and CL4 with 64 members,Chart 2 and Chart 3). Another special example is the small 10-membersublibrary CL2. Since fluorinated compounds may possess enhancedactivities and since fluorine has similar atomic radius with hydrogen,CL2 (10 members, Chart 2) was constructed with fluorine-substitutedphenethylamines and phenethylamine.

CL2 is significantly more potent than CL3 and CL4. However, at thisstage, we could not exclude the possibility that dilution in CL3 and CIAplayed a function since they contain more members. Thus, one of thesetwo sublibraries, CL3, was split to CL5 (28 members) and CL6 (27members, Chart 4). CL6 displayed higher potency, being split to CL7 (15members) and CL8 (14 members, two members were overlapping in CL7 andCL8). Library CL8 is more active in the MTT assay, it was split to CL11(6 members) and CL12 (8 members). The eight members in CL12 weresynthesized individually. At this stage, since the difference betweenCL8 and CL7 was not significant, CL7 was also split to CL9 and CL10. Andthe members of CL10 were synthesized individually. The splitting processis summarized in Chart 3. The activity data are shown in Table 1. Themost active CL10 compound was CL10b. The most active CL12 compounds wereCL12a and CL12b. TABLE 1 Structure and Activity of Libraries CL2, CL10and CL12. IC₅₀ Structure R^(i) R^(j) MW Nalm6 (MTT) Molt3 CL1 13.1 37.7CL2 0.9 3.7 CL2a H H 284.4 18.2 17.4 CL2b H 2-F 302.4 21.3 21.1 CL2c H3-F 302.4 17.6 12.1 CL2d H 4-F 302.4 17.5 13.8 CL2e 2-F 2-F 320.4 19.819.8 CL2f 2-F 3-F 320.4 17.0 13.0 CL2g 2-F 4-F 320.4 13.9 11.2 CL2h 3-F3-F 320.4 14.7 12.7 CL2i 3-F 4-F 320.4 18.2 12.9 CL2j 4-F 4-F 320.4unstable compound CL3 92.5 52.6 CL4 84.0 98.8 CL5 21.4 32.4 CL6 4.9 10.0CL7 18.7 13.3 CL8 7.7 8.4 CL9 45.6 50.0 CL10 26.2 26.2 CL10a 4-F2,5-(MeO)₂ 362.5 29.2 28.5 CL10b 4-F 2,4-Cl₂ 371.3 14.8 26.3 CL10c 4-Cl₂2,5-(MeO)₂ 378.9 33.6 29.0 CL10d 4-Cl 2,4-Cl₂ 487.8 22.0 29.7 CL10e 4-F3,4-(BnO)₂ 514.7 >100 >51.4 CL10f 4-Cl 3,4-(BnO)₂ 531.1 >100 >100 CL1171.6 42.7 CL12a 2-MeO 2,5-(MeO)₂ 374.5 17.4 10.9 CL12b 2-MeO 2,4-Cl₂383.3 17.1 11.6 CL12c 2,5-(MeO)₂ 2,5-(MeO)₂ 404.5 28.9 39.8 CL12d2,5-(MeO)₂ 2,4-Cl₂ 413.4 40.9 34.1 CL12e 2-MeO 3,4-(BnO)₂ 526.7 58.176.7 CL12f 2,5-(MeO)₂ 3,4-(BnO)₂ 556.7 27.4 37.2 CL12g 2,4-Cl₂3,4-(BnO)₂ 565.6 >100 21.8 CL12h 3,4-(BnO)₂ 3,4-(BnO)₂ 708.9 47.8 73.3Cytotoxic Activity Assay

The cytotoxic activity of the CL2 library compounds was investigated viaapoptosis/TUNEL assay. TUNEL assay allows the detection of exposed 3hydroxyl groups in fragmented DNA. The CL2 compounds prepared asdescribed above, were tested, along with DMSO as a control.

The cytotoxicity assay of various CL2 compounds against human tumor celllines was performed using the MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay(Boehringer Mannheim Corp., Indianapolis, Ind.). Briefly, exponentiallygrowing tumor cells were seeded into a 96-well plate at a density of2.5×10⁴ cells/well and incubated for 36 hours at 37° C. prior tocompound exposure. On the day of treatment, culture medium was carefullyaspirated from the wells and replaced with fresh medium containing theCL2 compounds at concentrations ranging from 0.0 to 100 μM. Triplicatewells were used for each treatment.

Human cell lines were obtained from American Type Culture Collection(Rockville, Md.) and maintained as a continuous cell line in Dulbecco'smodified Eagles' medium supplemented with 10% fetal bovine serum andantibiotics. Cells used in this study include human leukemia cells(NALM-6 and MOLT-3).

The cells were incubated with the various compounds for 24-36 hours at37° C. in a humidified 5% CO₂ atmosphere. To each well, 10 μl of MTT(0.5 mg/ml final concentration) was added and the plates were incubatedat 37° C. for 4 hours to allow MTT to form formazan crystals by reactingwith metabolically active cells. The formazan crystals were solubilizedovernight at 37° C. in a solution containing 10% SDS in 0.01 M HCl. Theabsorbance of each well was measured in a microplate reader (Labsystems)at 540 nm and a reference wavelength of 690 nm. To translate the OD₅₄₀values into the number of live cells in each well, the OD₅₄₀ values werecompared to those on standard OD₅₄₀—versus—cell number curves generatedfor each cell line. The percent survival was calculated using theformula:${\%\quad{Survival}} = {\frac{{live}\quad{cell}\quad{{number}\quad\lbrack{test}\rbrack}}{{live}\quad{cell}\quad{{number}\quad\lbrack{control}\rbrack}} \times 100}$

The IC₅₀ values were calculated by non-linear regression analysis.

The demonstration of apoptosis was performed by the in situnick-end-labeling method using ApopTag in situ detection kit (Oncor,Gaithersburg, Md.) according to the manufacturer's recommendations.Exponentially growing cells were seeded in 6-well tissue culture platesat a density of 50×10⁴ cells/well and cultured for 36 hours at 37° C. ina humidified 5% CO₂ atmosphere. The supernatant culture medium wascarefully aspirated and replaced with fresh medium containingunconjugated EGF or EGF-P154 at a concentration of 10, 25, or 50 Tg/ml.After a 36 hour incubation at 37° C. in a humidified 5% CO₂ incubator,the supernatants were carefully aspirated and the cells were treated for1-2 minutes with 0.1% trypsin. The detached cells were collected into a15 ml centrifuge tube, washed with medium and pelleted by centrifugationat 1000 rpm for 5 minutes. Cells were resuspended in 50 Tl of PBS,transferred to poly-L-lysine coated coverslips and allowed to attach for15 minutes. The cells were washed once with PBS and incubated withequilibration buffer for 10 minutes at room temperature.

After removal of the equilibration buffer, cells were incubated for 1hour at 37° C. with the reaction mixture containing terminaldeoxynucleotidyl transferase (TdT) and digoxigenin-11-UTP for labelingof exposed 3′-hydroxyl ends of fragmented nuclear DNA. The cells werewashed with PBS and incubated with anti-digoxigenin antibody conjugatedto FITC for 1 hour at room temperature to detect the incorporated dUTP.After washing the cells with PBS, the coverslips were mounted ontoslides with Vectashield containing propidium iodide (Vector Labs,Burlingame, Calif.) and viewed with a confocal laser scanningmicroscope. Non-apoptotic cells do not incorporate significant amountsof dUTP due to lack of exposed 3-hydroxyl ends, and consequently havemuch less fluorescence than apoptotic cells which have an abundance ofexposed 3′-hydroxyl ends. In control reactions, the TdT enzyme wasomitted from the reaction mixture.

The cytotoxic activities of CL1 and CL3 library compounds were alsoinvestigated via apoptosis/TUNEL assay described above. Combinatoriallibraries 1, 2, and 3 all caused apoptosis of NALM-6 leukemia cells in aconcentration-dependent fashion.

Example 2 Synthesis of Combinatorial Thiourea Mixture Libraries inSolution Phase

A mixture containing 1830 components was synthesized in solution phasein one step from thiocarbonyldiimidazole and 60 commercially-availableamines. Subsequently, 60 positional scanning deconvolution librarieswere synthesized for the identification of the active components.Several individual compounds were synthesized and tested for biologicactivity. Among them, 1,3-bis(1,2-diphenylethyl)-2-thiourea and1-(1,2-diphenylethyl)-3-(diphenylmethyl)-2-thiourea are the most potentwith IC₅₀ values below 10 μg/ml in MTT assays.

The parent library containing 1830 members was assembled in onesynthetic step using Scheme 3. The number of the members in CL34 iscalculated as: 60+C(60,2)=60+60!/(2×58!)=60+60×59/2=1830.

A mixture of equimolar 60 amines (Table 2) substituted the imidazolemoiety in TCDI under reflux. Specifically, a solution of 60 amines (1mmol each, Table 2) in acetonitrile (60 mL) was added into a solution ofTCDI (5.35 g, 30 mmol) in acetonitrile (100 mL) dropwise at 0° C.,followed by reflux for 1 h. The completion of the reaction was monitoredwith TLC. The same work-up was followed as described in Example 1. Ayellow gel was obtained as the desired library in 70% yield. ¹H NMRincluded all peaks observable with the individual compound and SML.TABLE 2 The 60 building blocks used to assemble the parent library.

A1

A2

A3

A4

A5

A6

A7

A8

A9

A10

A11

A12

A13

A14

A15

A16

A17

A18

A19

A20

A21

A22

A23

A24

A25

A26

A27

A28

A29

A30

A31

A32

A33

A34

A35

A36

A37

A38

A39

A40

A41

A42

A43

A44

A45

A46

A47

A48

A49

A50

A51

A52

A53

A54

A55

A56

A57

A58

A59

A60

The product mixture was purified by aqueous acid wash/extraction. Thereaction and purification conditions employed were the same as describedfor SML. The collected yield was 70%. The ESI-MS spectrum is shown inFIG. 2A and a computer-generated MS spectrum is shown in FIG. 2B forcomparison.

The IC₅₀ value of the CL34 parent library in MTT assays was 70 μg/ml.After the parent mixture library was discovered to be active, 60positional scanning deconvolution libraries (Boger, D. L.; Jiang, W.;Goldberg, J. J. Org. Chem. 1999, 64, 7094-7100; (b) Dooley, C. T.;Houghten, R. A. Life Sci. 1993, 52, 1509; (c) Pinilla, C.; Appel, J. R.;Blanc, P.; Houghten, R. A. Biotechniques, 1992, 13, 901) were assembledin order to identify those building blocks essential for an activethiourea library as shown in Scheme 4.

Each of the sublibraries is comprised of thiourea compounds that sharean identical N substitution on one end and differ from each other by thevariant N′ substitution on the other end representing one of the 60different building blocks shown in Table 2.

The solution phase combinatorial synthesis was accomplished using theArgonaut Quest 205 synthesizer, which can carry out 10 reactions inparallel. All of the reaction conditions used for glass flasks werereadily adapted for the synthesizer, except reflux was replaced withtemperature control at 81° C. (bp of acetonitrile) in sealed reactionvessels. Specifically, anhydrous acetonitrile (20 mL for each reactionvessel) was loaded in parallel in reaction vessels of Quest 205synthesizer, which consists of 10 reaction vessels with 100-mL capacityeach, followed by loading of TCDI (1.069 g, 6 mmol each reactionvessel). The reaction vessels were cooled down to 5° C. with circulatingice-water. Amines A1-A10 (Table 2) were dissolved in 20-mL anhydrousacetonitrile each separately, and added into the TCDI solutionsdropwise, separately. The reaction vessels were heated up and maintainedat 81° C. for 1 h in closed nitrogen-gas environment. The completion ofthe first substitution can be monitored with TLC. A stock solution ofamines A1-A60 (Table 2) (1 mmol each) were dissolved in 200-mLacetonitrile. The amine mixture solution (20 mL each time) was addedinto every reaction vessel sequentially via syringes. The reactionvessels were heated up and maintained at 81° C. for 1 h. The samework-up was followed as in Example 1. A yellow gel was obtained with a68-100% yield.

The biologic activity of the sublibraries was examined in standard MTTassays. The results, expressed as the IC₅₀ values (in μg/ml) are shownin Table 3. TABLE 3 MTT Assay Results and Yields of Synthesis of theDeconvolution Libraries IC₅₀, μg/mL Sublibrary Nalm6 Molt3 Yield, %CL34A1 41.05 51.2 94 CL34A2 5.6 63.8 96 CL34A3 8.7 28.4 100 CL34A4 7.926.3 100 CL34A5 19.0 8.6 92 CL34A6 15.7 41.9 100 CL34A7 52.9 45.4 93CL34A8 48.3 39.1 100 CL34A9 17.8 22.2 100 CL34A10 31.6 29.3 78 CL34A1138.3 35.6 81 CL34A12 33.4 49.6 91 CL34A13 43.7 42.0 100 CL34A14 78.672.5 81 CL34A15 76.3 95.7 83 CL34A16 78.6 97.4 84 CL34A17 41.3 76.8 83CL34A18 74.2 46.2 78 CL34A19 41.5 40.5 73 CL34A20 33.7 40.6 61 CL34A2137.0 39.1 100 CL34A22 38.6 45.9 93 CL34A23 60.2 98.5 91 CL34A24 92.185.3 92 CL34A25 44.7 29.2 84 CL34A26 56.3 >100 91 CL34A27 25.9 20.5 90CL34A28 55.5 50.3 92 CL34A29 44.2 45.6 71 CL34A30 58.6 >100 94 CL34A3156.3 >100 89 CL34A32 53.4 >100 92 CL34A33 44.8 64.5 89 CL34A34 54.4 >10095 CL34A35 93.1 99.3 99 CL34A36 62.1 >100 90 CL34A37 45.3 >100 96CL34A38 61.6 46.3 93 CL34A39 49.5 53.6 94 CL34A40 36.4 >100 84 CL34A4173.2 >100 91 CL34A42 78.3 >100 99 CL34A43 84.8 98.4 100 CL34A44 70.391.6 98 CL34A45 20.8 21.0 94 CL34A46 88.4 89.6 78 CL34A47 >100 >100 68CL34A48 89.6 83.6 78 CL34A49 97.4 95.6 93 CL34A50 91.9 >100 90 CL34A5195.6 >100 31 CL34A52 78.4 89.8 85 CL34A53 67.8 88.2 89 CL34A54 70.8 71.688 CL34A55 75.9 85.1 91 CL34A56 10.6 32.0 99 CL34A57 11.6 15.6 64CL34A58 48.3 91.5 98 CL34A59 62.1 71.5 86 CL34A60 63.3 57.6 85

The results in Table 2 indicate that sublibraries CL34A2, CL34A3,CL34A4, and CL34A5, containing 60 compounds each, have significantcytotoxic activity with IC50 values <10 μg/ml. Sublibraries CL34A56 andCL34A57 were also cytotoxic, with slightly higher IC50 values. 21individual compounds (6 symmetrical and 15 asymmetrical compounds) usingthe active building blocks of these 6 sublibraries were synthesized(Chart 4).

Specifically, anhydrous acetonitrile (10 mL for each reaction vessel)was loaded in parallel in reaction vessels of Quest 205 synthesizer,followed by loading of TCDI (0.535 g, 3 mmol each reaction vessel). Thereaction vessels were cooled down to 5° C. with circulating ice-water.Amines A2, A3, A5, A56 and A57 (6 mmol each) were dissolved in 20-mLanhydrous acetonitrile each separately, and added into #1-#5 of the TCDIsolutions, separately. Amine A2 (3 mmol each in three round-bottomedflasks) was dissolved in anhydrous acetonitrile (10-mL each) separately,and added into TCDI solutions in reaction vessels #6-#8 dropwise at 5°C. Amine A3 (3 mmol each in two round-bottomed flasks) was dissolved inanhydrous acetonitrile (10-mL each) separately, and added into TCDIsolutions in reaction vessels #9 and #10 dropwise at 5° C. The reactionvessels #6-#10 were heated up to and maintained at 81° C. for one hour(The temperature of the two banks, #1-#5 and #6-#10, can be controlledseparately). Amines A3, A4 and A5 were dissolved in 10-mL acetonitrileeach in three round-bottomed flasks, and added into reaction vessels#6-#8 dropwise at 5° C. Amines A4 and A5 were dissolved in 10-mLacetonitrile each in two round-bottomed flasks, and added into reactionvessels #9 and #10 separately at 5° C. The reaction vessels were heatedup to and maintained at 81° C. for one hour. The same work-up wasfollowed as in Example 1.

These 21 compounds were tested for cytotoxic activity against Nalm6leukemia cell line using MTT assays. The results are presented in Table4. The most active compounds were L34ASA5(1,3-bis(1,2-diphenylethyl)-2-thiourea) and L34A3AS(1-(1,2-diphenylethyl)-3-(diphenylmethyl)-2-thiourea). L34A2A5 andL34A3A56 ranked second best. TABLE 4 MTT Assay Results and Yields forthe Lead Compounds ED₅₀, μg/mL Compounds Nalm6 Yield, % Group 1 L34A2A250 76 L34A3A3 >100 78 L34A4A4 30 67 L34A5A5 <10 68 L34A2A3 50 67 L34A2A450 80 L34A2A5 25 68 L34A3A4 >50 82 L34A3A5 <10 85 L34A4A5 50 97 Group 2L34A56A56 50 78 L34A57A57 30 75 L34A2A56 >50 85 L34A2A57 >50 82 L34A3A5625 82 L34A3A57 30 81 L34A4A56 50 72 L34A4A57 30 63 L34A5A56 50 82L34A5A57 30 82 L34A56A57 >50 92

The physicochemical data for the two most active compounds are asfollows:

1,3-Bis(1,2-diphenylethyl)-2-thiourea (L34A5A5). A white powder (0.887g, 68% yield) was obtained as the desired product. ¹H NMR (CDCl₃, 300MHz) d 7.31-6.80 (m, 20H), 6.08 (bs, 2H), 5.08 (bs, 2H), 3.00 (d, J=6.6Hz, 4H); ¹³C NMR (CDCl₃, 75 MHz) d 180., 140.0, 136.1, 129.3, 129.1,128.7, 128.4, 128.2, 126.5, 126.2, 107.2, 60.0, 59.3, 43.1; m/z(MALDI-TOF) 437.1 (C₂₉H₂₈N₂S+H⁺ requires 437.6); UV-vis l_(max) 202,208, 253 nm; HPLC retention time 37.7 min, purity 98%.

1-(1,2-Diphenylethyl)-3-(diphenylmethyl)-2-thiourea (L34A3A5). A whitepowder (0.981 g, 77% yield) was obtained as the desired product. ¹H NMR(CDCl₃, 300 MHz) d 7.35-7.15 (m, 15H), 6.91 (m, 5H), 6.16 (m, 3H), 5.22(bs, 1H), 3.04 (m, 2H); m/z (MALDI-TOF) 422.4 (C₂₈H₂₆N₂S⁺ requires422.6); UV-vis l_(max) 202, 206, 253 nm; HPLC retention time 33.3 min,purity 92%.

Example 3 Synthesis of Urea Mixture Libraries

A mixture containing 1830 urea compounds was synthesized in one stepdepicted in Scheme 5 from carbonyldiimidazole and 60commercially-available amines shown in FIG. 3, as described for thioureacompounds in Example 2. Sixty positional scanning deconvolutionlibraries were synthesized for the identification of active components.FIG. 3A shows the ESI-MS spectrum of CL35. FIG. 3B shows acomputer-generated MS spectrum of CL35 for comparison.

The MTT assay results for these sublibraries are shown in Table 5. TABLE5 MTT Assay Results of the Deconvolution Libraries IC₅₀, μg/mLSublibrary Nalm6 Molt3 CL35A1 >100 >100 CL35A2 >100 >100CL35A3 >100 >100 CL35A4 36.9 24.1 CL35A5 20.6 22.9 CL35A6 33.3 26.2CL35A7 95.6 >100 CL35A8 90.2 91.3 CL35A9 37.9 31.8 CL35A10 81.4 96.4CL35A11 65.3 95.1 CL35A12 74.4 57.9 CL35A13 24.8 36.7 CL35A14 60.3 64.1CL35A15 86.2 >100 CL35A16 81.5 >100 CL35A17 71.2 96.1 CL35A18 84.2 90.5CL35A19 81.2 95.6 CL35A20 62.5 68.2 CL35A21 70.1 66.3 CL35A22 68.3 66.2CL35A23 88.2 81.5 CL35A24 70.6 95.4 CL35A25 58.1 56.2 CL35A26 60.4 66.2CL35A27 23.4 23.6 CL35A28 45.8 71.5 CL35A29 47.8 50.3 CL35A30 85.6 >100CL35A31 70.2 62.5 CL35A32 66.3 60.5 CL35A33 48.1 60.2 CL35A34 37.2 46.5CL35A35 66.3 >100 CL35A36 90.5 91.2 CL35A37 49.6 96.5 CL35A38 42.5 70.2CL35A39 36.2 48.9 CL35A40 70.2 94.0 CL35A41 49.2 37.9 CL35A42 70.5 78.2CL35A43 75.6 79.1 CL35A44 73.6 70.5 CL35A45 44.7 32.3 CL35A46 53.2 55.8CL35A47 >100 >100 CL35A48 >100 >100 CL35A49 98.1 92.6 CL35A50 96.8 90.2CL35A51 18.9 38.3 CL35A52 25.8 48.3 CL35A53 27.1 29.7 CL35A54 62.5 60.4CL35A55 56.8 50.6 CL35A56 53.4 55.9 CL35A57 42.6 52.6 CL35A58 43.4 40.2CL35A59 66.8 70.2 CL35A60 >100 >100Sublibraries CL35A2, CL35A3, CL35A4, and CL35A5, containing 60 compoundseach, were the most biologically active.

The present invention should not be considered limited to the particularexamples described above, but rather should be understood to cover allaspects of the invention as fairly set out in the attached claims.Various modifications, equivalent processes, as well as numerousstructures to which the present invention may be applicable will bereadily apparent to those of skill in the art to which the presentinvention is directed upon review of the instant specification.

The content of all publications, patents, and patent documents describedand cited herein is incorporated by reference as if fully set forth. Theinvention described herein may be modified to include alternativeembodiments. All such obvious alternatives are within the spirit andscope of the invention, as claimed below.

1. A process comprising: reacting a compound of formula I:

with an amine selected from:


2. A process comprising: reacting a compound of formula I:

with amines of formula:


3. A process comprising: reacting a compound of formula I:

with an amine selected from:


4. A process comprising: reacting a compound of formula I:

with amines of formula:


5. A product produced by the process of claim
 2. 6. The product of claim5, wherein the product includes a library of 1830 members.
 7. A productproduced by the process of claim
 4. 8. The product of claim 7, whereinthe product includes a library of 1830 members. 9 A compound selectedfrom the group of:


10. A method of killing a cancer cell by contacting the cancer cell withthe product of claim
 5. 11. A method of killing a cancer cell bycontacting the cancer cell with the product of claim
 7. 12. A methodcomprising administering to a cancer patient an effective cancer cellkilling amount of a compound of Formula I

wherein X is S or O; R and R₁ are individually

wherein Ar is aryl; R₂ is H or C₁ to C₆ alkyl; n is 0-3 and where thearyl moiety is optionally substituted from 1 to 7 times with anycombination of H, halo, alkyl, haloalkyl, arylalkyl, alkoxy, haloalkoxy,and aralkoxy.
 13. The method of claim 12, wherein R and R₁ areindividually selected from the group of:


14. The method of claim 12, wherein the compound is:


14. The method of claim 12, wherein the cancer patient has leukemia. 15.The method of claim 10, wherein the cancer cell is a leukemia cell. 16.The method of claim 11, wherein the cancer cell is a leukemia cell. 17.A kit for determining possible apoptosis induction agents for abiological substrate comprising the product of claim
 5. 18. A kit fordetermining possible apoptosis induction agents for a biologicalsubstrate comprising the product of claim 7.